Regulatory proteins serve as integrators of inter-and intracellular signals. Cellular information is often communicated in a language of posttranslational protein modification. This theme has been particularly apparent in cellular signaling pathways that impinge on the transcriptional program coordinated in the nucleus of the cell. Protein acetylation has been known for many years, but our knowledge of proteins regulated by acetylation is considerably less than for protein phosphorylation. Recent studies indicate that several key transcriptional regulators are regulated by reversible acetylation, which underscores the need to better understand the relevant acetylation pathways in the cell, and to link these events to specific regulatory functions. Dissecting these circuits will hopefully lead to a better understanding and treatment of diabetes, cancer and other human diseases. We are developing a chemical genetic strategy to identify cellular substrates of GCN5 and PCAF, two human Histone Acetyltransferase (HATs). These enzymes belong to the highly conserved GNAT family of HATs. We have successfully engineered the GNAT active site to allow the acetyl coenzyme A (CoA) binding pocket to accommodate unnatural acyl CoAs. We generated a library of synthetic acyl CoAs and screened them against the engineered GNAT domain to identify unique enzyme cofactor pairs. This approach yielded a GNAT mutant that catalyzes acyl transfer from from an unnatural CoA conjugate to histone tail peptides and nucleosomal substrates. Importantly, this acyl CoA is not a cofactor for wild-type GNAT domain. Thus, the engineered HAT covalently and specifically modifies substrates with a chemically unique tag. We are now utilizing click chemistry to facilitate direct visualization and affinity purification of novel substrates for two members of the GNAT family. Enrichment of specific substrates of the GNAT enzymes will enable protein identification using mass spectrometric techniques. We are pursing this approach in mammalian tissue culture cells and a variety of eukaryotic model organisms.